Department of Cellular and Molecular Physiology, College of Medicine, Penn State University, Hershey, Pennsylvania.
Am J Physiol Heart Circ Physiol. 2022 Jan 1;322(1):H71-H86. doi: 10.1152/ajpheart.00478.2021. Epub 2021 Nov 12.
Microvessels-on-a-chip have enabled in vitro studies to closely simulate in vivo microvessel environment. However, assessing microvessel permeability, a functional measure of microvascular exchange, has not been attainable in nonpermeable microfluidic platforms. This study developed a new approach that enables permeability coefficients () to be quantified in microvessels developed in nonpermeable chip platforms by integrating avidin-biotin technology. Microvessels were developed on biotinylated fibronectin-coated microfluidic channels. Solute transport was assessed by perfusing microvessels with fluorescence-labeled avidin. Avidin molecules that crossed endothelium were captured by substrate biotin and recorded with real-time confocal images. The was derived from the rate of avidin-biotin accumulation at the substrate relative to solute concentration difference across microvessel wall. Avidin tracers with different physiochemical properties were used to characterize the barrier properties of the microvessel wall. The measured baseline and inflammatory mediator-induced increases in and endothelial cell (EC) [Ca] resembled those observed in intact microvessels. Importantly, the spatial accumulation of avidin-biotin at substrate defines the transport pathways. Glycocalyx layer is well formed on endothelium and its degradation increased transcellular transport without affecting EC junctions. This study demonstrated that in vitro microvessels developed in this simply designed microfluidics structurally possess in vivo-like glycocalyx layer and EC junctions and functionally recapitulate basal barrier properties and stimuli-induced responses observed in intact microvessels. This new approach overcomes the limitations of nonpermeable microfluidics and provides an easily executed highly reproducible in vitro microvessel model with in vivo microvessel functionality, suitable for a wide range of applications in blood and vascular research and drug development. Our study developed a novel method that allows permeability coefficient to be measured in microvessels developed in nonpermeable microfluidic platforms using avidin-biotin technology. It overcomes the major limitation of nonpermeable microfluidic system and provides a simply designed easily executed and highly reproducible in vitro microvessel model with permeability accessibility. This model with in vivo-like endothelial junctions, glycocalyx, and permeability properties advances microfluidics in microvascular research, suitable for a wide range of biomedical and clinical applications.
微血管芯片使体外研究能够更紧密地模拟体内微血管环境。然而，在不可渗透的微流控平台中，评估微血管通透性（一种微血管交换的功能测量）是无法实现的。本研究开发了一种新方法，通过整合亲和素-生物素技术，可以在不可渗透的芯片平台上开发的微血管中定量测量渗透系数（）。微血管在生物素化纤维连接蛋白涂覆的微流控通道上形成。通过用荧光标记的亲和素来灌注微血管来评估溶质转运。穿过内皮的亲和素分子被基质生物素捕获，并通过实时共焦图像记录下来。是通过相对于微血管壁两侧溶质浓度差的基质中亲和素-生物素积累速率来推导的。使用具有不同物理化学性质的亲和素示踪剂来表征微血管壁的屏障特性。测量的基础和炎症介质诱导的和增加以及内皮细胞（EC）[Ca]与完整微血管中观察到的相似。重要的是，亲和素-生物素在基质上的空间积累定义了转运途径。内皮细胞上的糖萼层形成良好，其降解增加了细胞间转运，而不影响 EC 连接。本研究表明，在这种简单设计的微流控中体外形成的微血管在结构上具有类似于体内的糖萼层和 EC 连接，并且在功能上再现了完整微血管中观察到的基础屏障特性和刺激诱导的反应。这种新方法克服了不可渗透微流控的局限性，并提供了一种易于执行且高度可重复的具有体内微血管功能的体外微血管模型，适用于广泛的血液和血管研究和药物开发应用。我们的研究开发了一种新方法，使用亲和素-生物素技术在不可渗透的微流控平台上开发的微血管中测量渗透系数。它克服了不可渗透微流控系统的主要限制，并提供了一种简单设计、易于执行且高度可重复的具有渗透性的体外微血管模型。该模型具有类似于体内的内皮连接、糖萼和渗透性特性，推动了微流控在微血管研究中的发展，适用于广泛的生物医学和临床应用。
